Team:Paris Liliane Bettencourt/Notebook/2010/07/24/



</td></tr> <tr> <td style="background-color: #EEE"><a href="/wiki/Image:Igem-logo-150px.png" class="image" title="Igem-logo-150px.png"><img alt="" src="/images/7/7b/Igem-logo-150px.png" width="150" height="115" border="0" /></a> Population counter </td><td style="background-color: #F2F2F2" align="center"><img src="/images/9/94/Report.png" border="0" /> <a href="/wiki/IGEM:Paris_Liliane_Bettencourt/Notebook/Paris_2010" title="IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010">Main project page</a>
<img src="/images/c/c3/Resultset_previous.png" border="0" /><a href="/wiki/IGEM:Paris_Liliane_Bettencourt/Notebook/Paris_2010/2010/07/23" title="IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/23">Previous entry</a>      <a href="/wiki/IGEM:Paris_Liliane_Bettencourt/Notebook/Paris_2010/2010/07/25" title="IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/25">Next entry</a><img src="/images/5/5c/Resultset_next.png" border="0" /> </td></tr> <tr>

Minipreps : 4 minipreps of a 10ml culture per biobrick, eluted with 30μL buffer and collected into 1 tube for a total volume of around 120μL:

  • LacZ alpha (I732006)
  • RBS/GFP/term (E0240)
  • LuxR (C0062)
  • RBS/LuxI (K081008)
  • TetA(C) (J31007)

Restriction digest of the same minipreps as yestarday (concentration measured by nanodrop 07/23), around 3µg of DNA per reaction.

  • term/attC/mRFP : 42µl DNA + 5µl Buffer 3 10x + 0.5µl BSA 100x + 2µl XbaI + 2µl PstI
  • attC/term/attC/mRFP : 42µl DNA + 5µl Buffer 3 10x + 0.5µl BSA 100x + 2µl XbaI + 2µl PstI

Gel electrophoresis of the restriction digest products (future inserts)
Agarose gel 1.5%, 50V

  • Ladder 1Kb
  • attC/term/attC/mRFP : 25µl + 5µl lb
  • attC/term/attC/mRFP : 25µl + 5µl lb
  • -
  • term/attC/mRFP : 25µl + 5µl lb
  • term/attC/mRFP : 25µl + 5µl lb

<a href="/wiki/Image:Gel2407_1.jpg" class="image" title="Image:gel2407_1.jpg"><img alt="Image:gel2407_1.jpg" src="/images/2/21/Gel2407_1.jpg" width="357" height="409" border="0" /></a> <a href="/wiki/Image:Gel2407_2.jpg" class="image" title="Image:gel2407_2.jpg"><img alt="Image:gel2407_2.jpg" src="/images/f/fb/Gel2407_2.jpg" width="371" height="414" border="0" /></a>
The absence of the band corresponding to the insert for attC/term/attC/mRFP raises some questions about the real nature of this plasmid...

Gel purification with the Quiagen kit.

All bands are eluted with 30μL of water.

Overnight cultures of the transformations from 07/23 (colonies on all plates):

2x20mL for each transformation

  • TOP10 pSB1A2 weak RBS -TetA(C) - Amp
  • TOP10 pSB1A2 med.RBS -TetA(C) - Amp
  • TOP10 pSB1A2 weak RBS -LuxR - Amp
  • TOP10 pSB1A2 med.RBS -LuxR - Amp
  • TOP10 pSB1A2 stand.RBS -LuxR - Amp

Nanodrop to determine the amount of DNA in the miniprep samples and after gel purification:

Sample name 260/280 260/230 ng/μL
t-attC-mRFP (gel purif.) 1.610.026.3
LuxI (miniprep) 1.92 2.30203.4
LuxR (miniprep) 1.902.30221.5
LacZ (miniprep) 1.91 2.33 299.0
TetA(C) (miniprep) 1.91 2.26 303.4
GFP (miniprep) 1.90 2.29 239.1